Amides, C18 (unsaturated)

Neurotoxicity

Administrative data

Endpoint:

neurotoxicity

Remarks:

other:

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study well documented, meets generally accepted scientific principles, acceptable for assessment.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

The effects of oleamide on the performance of rats in tests for motor activity, analgesia, tolerance development, physical dependence and anxiety were investigated.

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

Oleamide

EC Number:

206-103-9

EC Name:

Oleamide

Cas Number:

301-02-0

Molecular formula:

C18H35NO

IUPAC Name:

octadec-9-enamide

Details on test material:

- Name of test material (as cited in study report): Oleamide
- Analytical purity: not reported

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic Laboratories, Frederick, MD, USA
- Weight at study initiation: 150-250g
- Housing: in an American Association for the Accreditation of Laboratory Animal Care-accredited facility

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

other: 5% DMSO/20% Alkamuls/75% water

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

Single treatments for determination of effects on motor activity, antinociception and thermoregulation. Application once daily for 3 days followed byapplication twice daily for 5 days for determination of tolerance to immobility, hypothermia, and analgesia. Application twice daily over 10 days for investigation of the potential of the test substance to induce physical dependence.

Frequency of treatment:

For determination of tolerance to immobility, hypothermia and analgesia 20 mg/kg once daily for the first 3 days followed by application of 30 mg/kg twice daily for 5 days. For investigation of the potential to induce physical dependence 30 mg/kg twice daily for 10 days.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
10-100 mg/kg i.p.
Basis:
nominal conc.
Determination of effects on motor activity, antinociception and thermoregulation.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
5 mg/kg i.p.
Basis:
nominal conc.
Effects on anxiety.

Doses / concentrations 3

Remarks:

Doses / Concentrations:
20 mg/kg i.p.
Basis:
nominal conc.
Determination of duration of test substance-induced effects.

Doses / concentrations 4

Remarks:

Doses / Concentrations:
20 mg/kg i.p once daily for 3 days, followed by 30 mg/kg i.p. twice daily for 5 days
Basis:
nominal conc.
Investigation of tolerance development to immobility, hypothermia, and analgesia.

Doses / concentrations 5

Remarks:

Doses / Concentrations:
30 mg/kg i.p. twice daily for 10 days
Basis:
nominal conc.
Studies of the potential of test substance to induce physical dependence.

No. of animals per sex per dose:

8 rats per dose for determination of effects on motor activity, antinociception, thermoregulation
9 rats per dose for determination of effects on anxiety
5 rats per dose for determination of duration of induced effects
6 rats per dose for determination of tolerance development towards test substance application
6 rats per dose for determination of physical dependence

Examinations

Neurobehavioural examinations performed and frequency:

MOTOR FUNCTION TESTS
OPEN FIELD PERFORMANCE
-Equipment: open field apparatus
-Parameters examined: distance travelled, number of vertical movements, number of stereotypic movements, time ambulating, time resting.
- Time schedule for examinations: after administration of the test compound. Parameters were monitored and recorded over 20 min.
CATALEPSY/INCLINED GRID TEST
-Equipment: inclined grid test
-Test procedure: Rats were placed on a 30 x 30 cm grid inclined 60°, and then the time the rat remained immobile measured for 2.5 min.
-Scoring criteria: the degree of catalepsy was scored from 0 to 5 based on the amount of the time the animal remained immobile (min): 0=0 to 0.08; 1= 0.09 to 0.35, 2=0.36 to 0.8, 3= 0.81 to 1.42; 4= 1.42 to 2.24; and 5 > 2.25 min

TEST OF ANALGESIA
TAIL-FLICK TEST
Test procedure: rat tails were placed under the focused beam of a halogen projection lamp. The intensity of the lamp was adjusted so that the unrestrained tail of a normal rat did not remain under the bean for longer than 4 s, with a cutoff time of 10 s. The latency to remove the tail from the beam path (s) following treatment was recorded and processed to yield the percentage of a maximum possible effect: % MPE= 100 X [(test-control)/(10-control)].
HOT-PLATE TEST
Rats were placed in a plastic cylinder atop a hot-plate uniformly regulated to 55°C over the entire surface. The amount of time before the rat showed evidence of thermal discomfort (e.g, licking a paw) was recorded and the test ended.

TEST OF AXIETY
SOCIAL INTERACTION TEST
-Equipment: a 1x 1 m² arena of white acrylic.
-Parameters (active social interactions) examined: sniffing, following, grooming, biting, boxing, and crawling over or under the cohort.
- Time schedule for examinations: the above mentioned parameters were measured over a 15 min period.
-Environment: The social interaction test was conducted under bright light in an unfamiliar environment.

ELEVATED PLUS-MAZE
-Equipment: elevated plus-maze composed of two open (790 x 15 cm) and two enclosed (70 x 15 x 15 cm) arms constructed of black acrylic radiating from a central platform to form a plus sign. The entire apparatus was elevated to a height of 1.2 m above the floor level by a single central support.
- Test procedure: Testing (5 min duration) was commenced by placing a rat on the central platform of the maze facing an open arm.
- Parameters: the number of open and closed arm entries (arm entry= all four paws into maze arm) and the time spent in various sections of the maze.

INDUCTION OF TOLERANCE/DEPENDENCE AND SCORING OF WITHDRAWAL SYMPTOMS
Tolerance to immobility, hypothermia, and analgesia were determined 20 mg/kg oleamide i.p, daily for 3 days, followed by administration of 30 mg/kg oleamide twice daily for the next 5 days. Studies of the potential of oleamide to induce physical dependence were performed after 10 days of administrating oleamide, 30 mg/kg twice daily, at 10:00 AM and 4:00 PM. Abstinence behaviours were measured over 1 h period after spontaneous withdrawal (observation starting 16 h after the last dose) or after withdrawal was precipitated with 4.5 mg/kg SR 141716 A. These behaviours included scratching, wet-dog shakes, head shakes, back arching, and teeth chattering. The occurrence of these behaviours was converted into an abstinence score reflecting the average number of times any withdrawal-associated behaviours were observed over a 1h period.

Results and discussion

Results of examinations

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Effects only temporary and not adverse, fully reversible; development of tolerance after repeated exposure and no development of physical dependence.

Details on results:

NEUROBEHAVIOUR
Oleamide (10-100 mg/kg i.p) significantly suppressed locomotion in the open field, as evidenced by a decrease in distance travelled with increasing dose.
Oleamide (10-200 mg/g) also exhibited analgesic activity, increasing the latency in the tail-flick test. Oleamide administration (10-100 mg/kg) resulted in hypothermia, but had no noticeable cataleptic actions as measured in the inclined grid test.
Finally, a dose of 5 mg/kg showed significant anxiolytic effects in both the social interaction test and plus-maze performance, increasing both the number of entries into the open arm, as well the time spent in the open arm.
The oleamide-induced (20 mg/kg) decrease in the distance travelled in the open field was maximal at 30 min after administration and lasted 60 min. The analgesic properties of oleamide were also maximal at 30 min and lasted for at least 1 h. In contrast, the hypothermic actions of oleamide, while maximal at 30 min, were gone by 60 min after administration i.p.
3 days of oleamide administration did not produce tolerance to any of its actions following a challenge dose [20 mg/kg, oleamide] except for hypothermia. In contrast, after 8 days of treatment, administration of a challenge dose of oleamide had no effect on the distance travelled in the open field, tail-flick latency, or body temperature.
Oleamide was further demonstrated to be a poor inducer of physical dependence; animals spontaneously withdrawn from oleamide after a 10-day period of application failed to show typical sings of withdrawal behaviour. 

Applicant's summary and conclusion

Conclusions:

No neurotoxic effects were observed.